The dopamine system is an extremely important system for essential motor regulation, hormone secretion regulation, emotion regulation, and such in the mammalian brain. Thus, abnormalities in dopaminergic neural transmission cause various neural disorders. For example, Parkinson's disease is a neurodegenerative disease of the extrapyramidal system that occurs due to specific degeneration of dopaminergic neurons in the substantia nigra of the midbrain (Harrison's Principles of Internal Medicine, Vol. 2, 23rd edition, Isselbacher et al., ed., McGraw-Hill Inc., NY (1994), pp. 2275-7). As a primary therapeutic method for Parkinson's disease, oral administration of L-DOPA (3,4-dihydroxyphenylalanine) is performed to compensate for the decrease in the amount of dopamine produced; however, the duration of the effect is known to be unsatisfactory.
More recently, a therapeutic method for Parkinson's disease was attempted in which the midbrain ventral regions of 6 to 9-week old aborted fetuses containing dopaminergic neuron progenitor cells are transplanted to compensate for the loss of dopaminergic neurons (Patent Document 1; and Non-Patent Documents 1 to 6). However, in addition to cell supply and ethical issues (Rosenstain (1995) Exp. Neurol. 33: 106; Turner et al. (1993) Neurosurg. 33: 1031-7), this method is currently under criticism for various other problems, including risk of infection and contamination, immunological rejection of transplants (Lopez-Lozano et al. (1997) Transp. Proc. 29: 977-980; Widner and Brudin (1988) Brain Res. Rev. 13: 287-324), and low survival rates due to the primary dependence of fetal tissues on lipid metabolism rather than glycolysis (Rosenstein (1995) Exp. Neurol. 33: 106).
In order to resolve the ethical issues and shortage of supply, methods have been proposed that use, for example, porcine cortex, stria, and midbrain cells (for example, see Patent Documents 2 to 4). In these methods, a complex procedure that involves altering cell surface antigens (MHC class I antigens) is required to suppress rejection. A method involving local immunosuppression by simultaneously transplanting Sertoli's cells has been proposed as a method for eliminating transplant rejection (Patent Documents 5 and 6; and Non-Patent Document 7). It is possible to obtain transplant cells from relatives that have matching MHCs, bone marrow from other individuals, bone marrow banks, or umbilical cord-blood banks. However, if it were possible to use the patient's own cells, the problem of rejection reactions could be overcome without any laborious procedures or trouble.
Therefore, as transplant materials, the use of dopaminergic neurons differentiated in vitro from non-neural cells such as embryonic stem (ES) cells and bone marrow interstitial cells, instead of cells from aborted fetuses, is considered to be promising. In fact, functional dopaminergic neurons were reported to have been formed by transplanting ES cells to lesion stria of a rat Parkinson's disease model (Non-Patent Document 8). It is believed that the importance of regenerative therapy from ES cells or the patient's own nerve stem cells will increase in the future.
In treating damaged nerve tissue, it is necessary to reconstruct brain function, and in order to form a suitable link with surrounding cells (network formation), it is necessary to transplant immature cells, cells capable of differentiating into neurons in vivo. In the transplanting of neuron progenitor cells, in addition to the aforementioned problem regarding supply, there is also the possibility that the progenitor cells will differentiate into groups of heterogeneous cells. For example, in treating Parkinson's disease, it is necessary to selectively transplant the catecholamine-containing neurons that produce dopamine. Examples of transplant cells that have previously been proposed for use in the treatment of Parkinson's disease include striatum (Non-Patent Documents 3 and 9), immortalized cell lines derived from human fetal neurons (Patent Documents 7 to 9), human postmitotic neurons derived from NT2Z cells (Patent Document 10), primordial neuron cells (Patent Document 11), cells and bone marrow stroma cells transfected with exogenous genes so as to produce catecholamines such as dopamines (Patent Documents 12 and 13), and genetically engineered ES cells (Non-Patent Document 8). Additionally, the use of dopaminergic neurons formed by contacting nerve progenitor cells derived from fetal midbrain tissue with FGF-8 and Shh (Patent Document 14), and of tyrosine hydroxylase-expressing cells obtained by treating NT2 nerve cells with retinoic acid (Patent Document 15) has been proposed. However, none of these contain only dopaminergic neurons or cells that differentiate into dopaminergic cells.
A method has been proposed for selectively concentrating and isolating dopaminergic neurons from undifferentiated cell populations. In this method, a reporter gene that expresses a fluorescent protein is introduced into each cell of a cell population, under the control of a gene promoter/enhancer such as the tyrosine hydroxylase expressed in dopaminergic neurons (hereinbelow, also referred to as “TH”), and then cells emitting fluorescence are isolated. The dopaminergic neurons are visualized in their viable state, then concentrated, isolated, and identified (Patent Document 16). This method requires the complicated step of introducing an exogenous gene, and further, the presence of a reporter gene poses problems of toxicity and immunogenicity when used in conjunction with gene therapy.    [Patent Document 1] U.S. Pat. No. 5,690,927    [Patent Document 2] Japanese Patent Kohyo Publication No. (JP-A) H10-508487 (unexamined Japanese national phase publication corresponding to a non-Japanese international publication)    [Patent Document 3] JP-A H10-508488    [Patent Document 4] JP-A H10-509034    [Patent Document 5] JP-A H11-509170    [Patent Document 6] JP-A H11-501818    [Patent Document 7] JP-A H8-509215    [Patent Document 8] JP-A H11-506930    [Patent Document 9] JP-A 2002-522070    [Patent Document 10] JP-A H9-5050554    [Patent Document 11] JP-A H11-509729    [Patent Document 12] JP-A 2002-504503    [Patent Document 13] JP-A 2002-513545    [Patent Document 14] U.S. Pat. No. 6,277,820    [Patent Document 15] International Publication WO 00/06700    [Patent Document 16] Japanese Patent Application Kokai Publication No. (JP-A) 2002-51775 (unexamined, published Japanese patent application)    [Non-Patent Document 1] Spencer et al. (1992) N. Engl. J. Med. 327: 1541-8    [Non-Patent Document 2] Freed et al. (1992) N. Engl. J. Med. 327: 1549-55    [Non-Patent Document 3] Widner et al. (1992) N. Engl. J. Med. 327: 1556-63    [Non-Patent Document 4] Kordower et al. (1995) N. Engl. J. Med. 332: 1118-24    [Non-Patent Document 5] Defer et al. (1996) Brain 119: 41-50    [Non-Patent Document 6] Lopez-Lozano et al. (1997) Transp. Proc. 29: 977-80    [Non-Patent Document 7] Selawry and Cameron (1993) Cell Transplant 2: 123-9    [Non-Patent Document 8] Kim et al. (2002) Nature 418: 50-56    [Non-Patent Document 9] Lindvall et al. (1989) Arch. Neurol. 46: 615-31